CN109239019A - The preparation and its application method of surface plasmon resonance sensing chip - Google Patents
The preparation and its application method of surface plasmon resonance sensing chip Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of surface plasmon resonance sensing chip, method includes the following steps: S1, cleaning and are dried at substrate, one layer of chromium film is prepared on substrate；S2, one layer of dielectric layer is prepared on chromium film；And S3, one layer of metal layer of formation on dielectric layer, metal layer includes layer gold or silver layer, wherein, dielectric layer includes poly- methyl-prop methyl formate, IR-140 dyestuff and methylene chloride, dielectric layer is used to improve the sensitivity of surface plasmon resonance sensing chip, and the detection range of enlarged surface plasma resonance sensing chip.The present invention also provides a kind of methods that the surface plasmon resonance sensing chip using above method preparation is detected.
Description
Technical field
The present invention relates to field of biological medicine more particularly to a kind of preparations of surface plasmon resonance sensing chip
Method and the method detected using surface plasmon resonance sensing chip prepared by the above method.
Background technique
Surface plasma body resonant vibration (Surface Plasmon Resonance, SPR) is occurred in planar metal membrane interface
A kind of physical optics phenomenon.SPR is very sensitive to the variations in refractive index near planar metal membrane interface.It can using SPR principle
To detect the interaction that the biomolecule in 100nm range above at the planar metal membrane interface occurs.SPR detection method has
High sensitivity is not necessarily to label, can get the advantages that kinetics process, thus can be used for many applications, but since gold detects
The concentration of small molecule analyte is not sensitive enough, it is therefore desirable to the resolution ratio of spr sensor is improved, so as to disease and cause of disease body-sensing
Dye carries out early stage reliable diagnosis and quickly analysis air and environmental pollution.
Half-peak breadth reduction, the variation of resonant angle or the variation of lambda1-wavelength of SPR spectroscopy curve, may make SPR
The resolution ratio of sensor is promoted.It is most of at present scientific since surface plasma excimer is generated in limited metal surface
Research has been devoted to reduce the half-peak breadth of SPR spectroscopy curve.For example, Doré metal method, remote SPR and waveguide coupling
The design patterns such as SPR (WCSPR), but the time needed for above-mentioned preparation method is longer, and the step of preparing is more complicated, it is difficult to
Realize mass production.
Summary of the invention
In order to overcome at least one aspect of the above problem, the embodiment of the present invention provides a kind of surface plasma body resonant vibration biography
The preparation of sense chip and application method can select one section of linearity in fixed point monitoring by preparing one layer of gain media layer
Very high section is detected, to improve the sensitivity of surface plasmon resonance sensing chip.
According to an aspect of the present invention, a kind of preparation method of surface plasmon resonance sensing chip, the party are provided
Method prepares one layer of chromium film the following steps are included: S1, cleaning and are dried at substrate on substrate；S2, one layer of medium is prepared on chromium film
Layer；And S3, one layer of metal layer of formation on dielectric layer, metal layer includes layer gold or silver layer, wherein dielectric layer includes poly- methyl
Third methyl formate, IR-140 dyestuff and methylene chloride, dielectric layer are used to improve the spirit of surface plasmon resonance sensing chip
Sensitivity, and the detection range of enlarged surface plasma resonance sensing chip.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, substrate are glass
Substrate.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, successively with dense sulphur
Glass substrate is washed in acid and deionization boiling.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, step S1 are used
The method of sputtering or deposition prepares chromium film, chromium film with a thickness of 3nm-10nm.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, step S2 packet
It includes: poly- methyl-prop methyl formate S2.1, being dissolved in methylene chloride, add IR-140 dyestuff and dielectric layer solution is made；And
S2.2, dielectric layer solution is uniformly dripped on substrate, obtains preliminary dielectric layer using spin-coating method, preliminary dielectric layer is toasted
Obtain dielectric layer.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, to preliminary medium
Layer carries out toasting to be included under 100 degrees Celsius toasting 5min.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, by dielectric layer
Solution further includes spin coating step after uniformly dripping on substrate, and the when a length of 15s of spin coating, the speed of spin coating is 500rpm.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, the rotation of spin-coating method
A length of 1min when painting, the range of spin speed are 3000rpm-5000rpm.
Some embodiments of the preparation method of surface plasmon resonance sensing chip according to the present invention, according to IR-140
The dielectric layer of different-thickness is made in the different ratio of dyestuff.
According to another aspect of the present invention, a kind of surface plasma resonance sensing prepared using the above method is provided
The method that chip is detected, method includes the following steps: Sa, by determinand buffer at the standard of various concentration
The standard solution of various concentration is mixed with the antibody of quantitative determinand respectively, obtains the mixed solution of various concentration by solution；
Sb, it is reacted respectively with surface plasmon resonance sensing chip with the mixed solution of various concentration, to surface plasma
The reaction zone of resonance sensing chip carries out fixed point monitoring, obtains the standard curve of each standard solution；And Sc, to surface etc. from
Daughter resonance sensing chip is handled, and next sample is continued to test.
Some embodiments of the method according to the present invention detected using surface plasmon resonance sensing chip, it is fixed
It selects monitoring to include the following steps: to irradiate buffer with light source, the incident angle of scanning light source, be absorbed peak curve；And
It selects an angle in linear region to be positioned in absorption peak curve, and is monitored.
Some embodiments of the method according to the present invention detected using surface plasmon resonance sensing chip,
The peak curve that is absorbed further includes later following steps: being handled with polynomial fitting data, be absorbed peak song
The matched curve of line.
Some embodiments of the method according to the present invention detected using surface plasmon resonance sensing chip are more
The fitting number of item formula fitting algorithm is 9 times -11 times.
Some embodiments of the method according to the present invention detected using surface plasmon resonance sensing chip, step
Further comprising the steps of before rapid Sa: SA, a series of glycerol for preparing concentration gradients successively use surface plasma resonance sensing
Chip is detected.
Compared with prior art, the present invention one of at least has the following advantages that:
(1) short preparation period can prepare completion in one day；
(2) preparation process is simple；
(3) the surface plasmon resonance sensing chip sensitivity prepared is higher.
Detailed description of the invention
By the description made for the present invention of below with reference to attached drawing, other objects and advantages of the present invention will be aobvious and easy
See, and can help that complete understanding of the invention will be obtained.
Fig. 1 is the flow chart of the preparation method of surface plasmon resonance sensing chip according to an embodiment of the present invention；
Fig. 2 is that the structure of the surface plasmon resonance sensing chip of preparation method preparation according to an embodiment of the present invention is shown
It is intended to；
Fig. 3 is the matched curve figure that polynomial fitting according to an embodiment of the present invention obtains；
Fig. 4 is the error of fitting figure that polynomial fitting according to an embodiment of the present invention obtains；
Fig. 5 is the standard curve of the gain media layer detection glycerol of different-thickness according to an embodiment of the present invention；
Fig. 6 is that the detection of the surface plasmon resonance sensing chip of preparation method preparation according to an embodiment of the present invention is sweet
Oil concentration gradient map.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The term used in embodiments of the present invention is only to be not intended to be limiting merely for for the purpose of describing particular embodiments
The present invention.Unless otherwise defined, the technical term or scientific term that the present invention uses should be tool in fields of the present invention
The ordinary meaning for thering is the personage of general technical ability to be understood.
The present invention provides preparation and application method and and the surface plasma of a kind of surface plasmon resonance sensing chip
The corresponding spr signal Processing Algorithm of resonance body sensing chip.The preparation side of surface plasmon resonance sensing chip of the invention
Period is shorter the time required to method, and preparation step simply prepare it is easy.Simultaneously provide signal processing algorithm be also easy to realize and
Be conducive to the promotion of surface plasmon resonance sensing chip sensitivity.
The embodiment of the present invention is further described with reference to the accompanying drawing.
Fig. 1 is the flow chart of the preparation method of surface plasmon resonance sensing chip according to an embodiment of the present invention.Such as
Shown in Fig. 1, the preparation method of surface plasmon resonance sensing chip includes the following steps:
S1, cleaning substrate are simultaneously dried, and one layer of chromium film is prepared on substrate.
Substrate can be glass substrate, successively wash glass substrate with the concentrated sulfuric acid and deionization boiling, then will boil after washing
Glass substrate is dried.Wherein, deionization boiling wash bags include following steps: submerging substrate using deionized water, be heated to 100
Degree Celsius to boil, boil and wash substrate 10min, the hydrotrope and bulky grain can be removed.It can be using sputtering or the method system of deposition
Standby chromium film, chromium film with a thickness of 3nm-10nm.
S2, one layer of dielectric layer is prepared on chromium film.The structure of this layer of dielectric layer is one layer of chemical film, by IR-140 and is gathered
Methyl-prop methyl formate is made.
According to preferred embodiment, step S2 the following steps are included:
S2.1, poly- methyl-prop methyl formate (PMMA) is dissolved in methylene chloride, adds IR-140 dyestuff and medium is made
Layer solution.Such as after 200mgPMMA being dissolved in methylene chloride, then IR-140 is separately added by with PMMA different specific weight
In above-mentioned obtained solution, gain media layer solution is obtained.
S2.2, dielectric layer solution is uniformly dripped on substrate, preliminary dielectric layer is obtained using spin-coating method, to preliminary dielectric layer
It is toasted to obtain dielectric layer.It further include spin coating step after uniformly dripping dielectric layer solution on substrate, when spin coating is a length of
15s, spin coating speed are 500rpm.Preliminary Jie can be obtained with the speed spin coating 1min of 3000rpm-5000rpm after spin coating
Preliminary dielectric layer is placed in 100 degrees Celsius of baking box and toasts 5min, can be prepared by medium layer film by matter layer.
Dielectric layer includes poly- methyl-prop methyl formate, IR-140 dyestuff and methylene chloride, and dielectric layer is for improving surface
The sensitivity of plasma resonance sensing chip, and the detection range of enlarged surface plasma resonance sensing chip.According to IR-
The dielectric layer of different-thickness can be made in the different ratio of 140 dyestuffs.
S3, one layer of metal layer is formed on dielectric layer, metal layer includes layer gold or silver layer.Using sputtering or the method for deposition
The golden film that a layer thickness is 400nm-500nm is prepared on dielectric layer, can be obtained gain surface plasma resonance sensing core
Piece.Golden film can be replaced with silverskin, also may include other metals in certain other embodiments.
Fig. 2 is that the structure of the surface plasmon resonance sensing chip of preparation method preparation according to an embodiment of the present invention is shown
It is intended to.As shown in Fig. 2, surface plasmon resonance sensing chip includes substrate 1, chromium film 2, dielectric layer 3 and metal layer 4.?
In certain possible embodiments, surface plasmon resonance sensing chip can also include the analysis liquid of setting on the metal layer
Layer, analysis liquid layer can be measured object solution, depending on specific experiment.
The surface plasmon resonance sensing chip as made from the above method can save 1 year, preferably in six months
It uses.
The application that the present invention also provides above-mentioned surface plasmon resonance sensing chips in field of biological medicine with
And a kind of improved signal processing algorithm.
The application include it is a kind of using the above method preparation surface plasmon resonance sensing chip detected
Method, method includes the following steps:
Sa, the standard solution that the standard items of determinand are configured to various concentration with PBS buffer solution, by the various concentration
Standard solution is mixed and is stood with the antibody of quantitative determinand small molecule respectively, obtains each standard solution and quantitative determinand is small
The mixed solution of the antibody of molecule.The concentration range of standard solution is generally 0.01~5000 μ g/L, preferably 0.1-1000 μ g/
L, more preferably 1-100 μ g/L.
Sb, it is reacted respectively with surface plasmon resonance sensing chip with the mixed solution of the various concentration, it is right
The reaction zone of the surface plasmon resonance sensing chip carries out fixed point monitoring, obtains the standard curve of each standard solution.
On the basis of PBS buffer solution, the mixed solution of each standard solution and quantitative determinand antibody is injected separately into surface
The micro-fluidic measuring cell of plasma resonance instrument occurs immune with the small molecule conjugate on surface plasmon resonance sensing chip
Reaction carries out fixed point monitoring to surface plasmon resonance sensing chip reaction zone, records the variation of spr signal, i.e. reflected light
The variation of (RU) by force obtains the surface plasma resonance kinetic curve of each standard solution.Above-mentioned quantitative determinand small molecule
Final concentration of the antibody in mixed solution be preferably 1mg/L-30mg/L, more preferably 10mg/L.
Fixed point monitoring includes the following steps:
Buffer is irradiated with light source, scans the incident angle of the light source, be absorbed peak curve.It is passed through PBS buffer solution
1min-5min, flow rates are then allowed to stand in 100 μ l/min-1000ml/min, then the incident angle of scanning light source, are obtained
SPR absorption peak curve, the range of angle scanning are 55 degree of -70 degree.
It selects an angle in linear region to be positioned in the absorption peak curve, and is monitored.With flow velocity
100 μ l/min-1000ml/min are passed through PBS buffer solution 1min-5min；Then it is passed through and is contained with 10 μ l/min-100ml/min of flow velocity
There is the mixed solution of sample to be tested and known antibodies concentration, mixed solution, which continuously flows, carries out Immune competition inhibition reaction 5min-
15min；Immune competition inhibits after reaction, to be passed through PBS buffer solution 1min- with 100 μ l/min-1000ml/min of flow velocity
5min；Then actified solution is passed through with 100 μ l/min-500ml/min of flow velocity, continuously flows 0.5min-1min；Regeneration ending
Afterwards, PBS buffer solution 1min-5min is passed through with 100 μ l/min-1000ml/min of flow velocity.The actified solution is preferably that pH is
HCl, the H of the Tris solution of 1.5-2.5 or concentration in 5mM-30mM3PO4Or NaOH solution.
Further include following steps after obtaining the absorption peak curve: with polynomial fitting to data at
Reason, obtains the matched curve of the absorption peak curve.For obtained SPR curve, using concentration of standard solution as abscissa,
Spr signal intensity draws working curve as ordinate, and with the improved polynomial fitting of the present invention to data at
Reason, makes matched curve；And small molecule is detected on this basis.
Steps are as follows for polynomial fitting:
Known one group of 2-D data, these data can be n different point (x in planei, yi), i=1,2,
3 ..., n finds a function y=f (x) and makes the function and all data points closest, i.e., fitting effect is best.More
In item formula fitting algorithm, f (x)=a is generally selected1xm+…+amx+am+1, wherein m is the order of fitting of a polynomial, in existing skill
In art, m is ordinarily selected to 2-7, uses 9-11 in the embodiment of the present invention, the effect of fitting is more preferable, and calculation amount is suitable.In above formula
Coefficient determined by least square method, formula specific as follows:
All coefficients can be acquired when J reaches minimum.Fig. 3 is that polynomial fitting according to an embodiment of the present invention obtains
Matched curve figure, according to Fig. 3, it can be seen that 11 ranks are fitted, passed through sample point is more and curve is also more smooth, therefore 11 ranks
SPR curve is better than 7 ranks.Fig. 4 is the error of fitting figure that polynomial fitting according to an embodiment of the present invention obtains.Such as Fig. 4 institute
Show, the error of fitting of 9-13 rank is significantly less than the fitting of 2-7 rank multinomial, illustrates that 9-13 rank is better than the fitting of 2-7 rank multinomial.
Sc, the surface plasmon resonance sensing chip is handled, continues to test next sample.Micro- flow cell
With 100 μ l/min~1000ml/min of flow velocity flowing be passed through PBS buffer solution cleaning 1min-5min, then with 100 μ l/min of flow velocity~
500ml/min is passed through actified solution and continuously flows 0.5min~1min；Dissociate antibody-antigen conjugates；Then with flow velocity 100
μ l/min~1000ml/min is passed through PBS buffer solution cleaning 1min-5min；When spr signal value drops back to baseline, continue to test next
A sample.
Before the step Sa further include: SA, a series of glycerol for preparing concentration gradients, successively with the surface plasma
Resonance body sensing chip is detected.Since the refractive index of various concentration glycerol is different, so being equipped with out a series of has concentration terraced
The glycerol of degree can detect the sensitivity of surface plasmon resonance sensing chip, namely verifying gain media layer film
Property.When experiment, 0-17 glycerol can be prepared, 0.5%, such as No. 0 is divided between percent concentration as water, No. 1 is 0.5%
Glycerol, it is 8.5% glycerol that No. 2, which are 1% glycerol ... 17,.Fig. 5 is the mark of the gain media layer detection glycerol of different-thickness
Directrix curve, wherein Fig. 5 (a), (b) abscissa indicate that angle pulse number, ordinate indicate light intensity.It can from Fig. 5
Out, by one layer of gain media layer of preparation, one section of very high section of the linearity can be selected to be detected in fixed point monitoring,
By caning be found that chip gain is better than traditional die after 11 rank multinomial fitting algorithms.Fig. 5 (b) is calculated by multinomial
The standard curve of the gain media layer detection glycerol of method treated different-thickness, by Fig. 5 (b) as can be seen that containing gain
The SPR chip of dielectric layer can have one section of very big linear zone of slope, therefore select this section that can obtain higher sensitivity.Fig. 6
It is the detection glycerol concentration gradient of the surface plasmon resonance sensing chip of preparation method preparation according to an embodiment of the present invention
Scheme, P indicates that the glycerine gradient of not chip gain, Q indicate the glycerine gradient of chip gain in Fig. 6.Abscissa represents in figure
Be the sampling time, ordinate is the light intensity of sampling.
Small molecule in the present embodiment can be folic acid, biotin, dopamine, small molecule pesticide, small molecule veterinary drug (such as sulphur
Amine, atrazine), micromolecule additive etc., it is all can be belonged to by the small molecule that conjugate generates antibody it is such.This implementation
Small molecule in example is preferably cortisol.
Chip gain prepared by the present invention has good SPR characteristic, not only has the advantages that the period is short, 1 day can
Preparation is completed, and preparation process is simple, with traditional SPR chip than available higher sensitivity.The present invention passes through system
Standby one layer of gain media layer, can select one section of very high section of the linearity to be detected in fixed point monitoring, more by 11 ranks
It can be found that chip gain is better than traditional die after item formula fitting algorithm.The present invention changes polynomial fitting
Into further improving the sensitivity of SPR chip.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (14)
1. a kind of preparation method of surface plasmon resonance sensing chip, which comprises the following steps:
S1, cleaning substrate are simultaneously dried, and prepare one layer of chromium film on the substrate；
S2, one layer of dielectric layer is prepared on the chromium film；And
S3, one layer of metal layer being formed on the dielectric layer, the metal layer includes layer gold or silver layer,
Wherein, the dielectric layer includes poly- methyl-prop methyl formate, IR-140 dyestuff and methylene chloride, and the dielectric layer is used for
The sensitivity of the surface plasmon resonance sensing chip is improved, and expands the surface plasmon resonance sensing chip
Detection range.
2. the preparation method of surface plasmon resonance sensing chip according to claim 1, which is characterized in that the base
Piece is glass substrate.
3. the preparation method of surface plasmon resonance sensing chip according to claim 2, which is characterized in that successively use
The glass substrate is washed in the concentrated sulfuric acid and deionization boiling.
4. the preparation method of surface plasmon resonance sensing chip according to claim 1, which is characterized in that step S1
Chromium film is prepared using the method for sputtering or deposition, the chromium film with a thickness of 3nm-10nm.
5. the preparation method of surface plasmon resonance sensing chip according to claim 1, which is characterized in that step S2
Include:
S2.1, poly- methyl-prop methyl formate is dissolved in methylene chloride, adds IR-140 dyestuff and dielectric layer solution is made；And
S2.2, the dielectric layer solution is uniformly dripped on the substrate, obtains preliminary dielectric layer using spin-coating method, to described first
Step dielectric layer is toasted to obtain dielectric layer.
6. the preparation method of surface plasmon resonance sensing chip according to claim 5, which is characterized in that described
Preliminary dielectric layer carries out toasting to be included under 100 degrees Celsius toasting 5min.
7. the preparation method of surface plasmon resonance sensing chip according to claim 5, which is characterized in that by institute
Stating after dielectric layer solution uniformly drips on the substrate further includes spin coating step, the when a length of 15s of spin coating, and the speed of spin coating is
500rpm。
8. the preparation method of surface plasmon resonance sensing chip according to claim 5, which is characterized in that the rotation
A length of 1min when the spin coating of coating, the range of spin speed are 3000rpm-5000rpm.
9. the preparation method of surface plasmon resonance sensing chip according to claim 1, which is characterized in that according to
The dielectric layer of different-thickness is made in the different ratio of IR-140 dyestuff.
10. a kind of surface plasma resonance sensing core prepared using method according to claim 1 to 9
The method that piece is detected, which comprises the following steps:
Sa, by determinand buffer at the standard solution of various concentration, the standard solution of the various concentration is distinguished
It is mixed with the antibody of quantitative determinand, obtains the mixed solution of various concentration；
Sb, it is reacted respectively with surface plasmon resonance sensing chip with the mixed solution of the various concentration, to described
The reaction zone of surface plasmon resonance sensing chip carries out fixed point monitoring, obtains the standard curve of each standard solution；And
Sc, the surface plasmon resonance sensing chip is handled, continues to test next sample.
11. according to the method described in claim 10, it is characterized in that, the fixed point monitoring includes the following steps:
Buffer is irradiated with light source, scans the incident angle of the light source, be absorbed peak curve；And
It selects an angle in linear region to be positioned in the absorption peak curve, and is monitored.
12. according to the method for claim 11, which is characterized in that after obtaining the absorption peak curve further include as follows
Step:
Data are handled with polynomial fitting, obtain the matched curve of the absorption peak curve.
13. according to the method for claim 12, which is characterized in that the fitting number of the polynomial fitting be 9 times-
11 times.
14. according to the method described in claim 10, it is characterized in that, further comprising the steps of before the step Sa:
SA, a series of glycerol for preparing concentration gradients, are successively detected with the surface plasmon resonance sensing chip.
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| CN106018347A (en) * | 2016-05-06 | 2016-10-12 | 中国科学院电子学研究所 | Surface plasma resonance sensing chip and preparation method and application thereof |
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| CN106018347A (en) * | 2016-05-06 | 2016-10-12 | 中国科学院电子学研究所 | Surface plasma resonance sensing chip and preparation method and application thereof |
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| AYDA ARAY等: "Gain-Assisted Surface Plasmon Resonance Refractive Index Sensor", 《IEEE SENSORS JOURNAL》 * |
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